PARTIAL CHARACTERIZATION OF EXOPEPTIDASES PRODUCED BY A STRAIN OF GEOTRICHUM-CANDIDUM

Amino and carboxypeptidase systems of Geotrichum candidum were investi gated in the strain P 25 being one of the ''felting'' biotype III, whi ch has the highest proteolytic activity in this species. It was grown in Czapeck-trypticase medium with initial pH 4, 5.5 or 7.5. The cultur e medium after mycelium separation by filtration was the exocellular p reparation. After washing and freezing, mycelium was disrupted in phos phate buffer either at pH 7.5 (aminopeptidase) or 5.5 (carboxypeptidas e), using ultra turrax then homogenizer with glass heads. This suspens ion represented crude mycelial extract and after it has been centrifug ed, the supernatant was defined as soluble mycelial extract. Activitie s were measured at pH 4, 6 and 7.5 on leucine p-nitro-anilide (Leu-p-N a) for aminopeptidases, and at pH 4, 5.5 and 7.5 on carbobenzoxy-gluta myl-tyrosine (Z-glu-tyr) and carbobenzoxy-glycyl-valine (Z-gly-val) fo r carboxypeptidases. Exocellular and soluble mycelial extracts were pa rtially purified on Sephadex G 100 after concentration by ultrafiltrat ion. Carboxypeptidase was further purified on DEAE Sephacel. The highe st levels of mycelial and exocellular aminopeptidase activities were o btained in static culture at pH 5.5, but they corresponded to two diff erent enzymatic systems. The optimal pH of mycelial system was 8-8.5, its maximal stability was in the pH range 5 to 9, its molecular weight about 85 000 and it was relatively heat-resistant. It seems due to on ly one enzyme which was particularly active on Leu-p-Na. On the other hand, exocellular system was relatively heat-labile, its molecular wei ght was about 150 000 and its maximum stability was at pH between 5.5 and 9.5. It hydrolysed preferably alanine-para-nitroanilide (Ala-p-Na) and, only at a Tower level, Leu-p-Na. It presented three optimal pH o f activity, one main at 7.5 and the another two at 6.5 and 9; its ther mal inactivation curve showed three slopes. These results suggest that exocellular aminopeptidase system contained probably at least three c omponents. Carboxypeptidase system, mainly mycelial bound or endocellu lar, seemed due to only one enzyme or to two closely related component s. Its optimal pH was about 4.5 on Z-glu-tyr and 5.5 on Z-gly-val, its maximum stability in the range of pH 5.5-6.5 and it preferably hydrol ysed Z-gly-val and Z-gly-leu at pH 5.5. Therefore, amino and carboxype ptidases from G. candidum are surely involved in cheese ripening, espe cially in soft cheese, where they could release free amino acids which are flavour precursors and also degrade potentially hitter peptides.